Substantial changes in CA2+ concentration ([CA2+]) in the fluid surrounding photoreceptors (subretinal space or SRS) follow light/dark transitions. Because Ca2+ affects photoreceptor function, SRS [Ca2+] must be tightly regulated. Though it is known that transport activity in the retinal pigment epithelium (RPE) subserves this regulatory role, the specific mechanisms have not been identified. All cells have only 2 mechanisms that can export Ca2+ across the plasma membrane: Na=:Ca2+ exchangers (NCXs) and plasma membrane Ca2+ ATPases (PMCAs). Except for studies in this application, neither of these proteins, nor the genes that encode them, have been characterized in human RPE. This project targets these two Ca2+ transport proteins. The fundamental hypothesis underlying planned studies is that the combined activities of a PMCA and NCX in RPE regulate light-dependent changes in SRS [Ca2+], and that impaired function of either of these Ca2+ transporters will cause impaired vision. Multiple genes and alternative splicing of primary mRNA transcripts govern the tissue-specific expression and regulation of PMCA and NCX proteins (termed isoforms). Thus, testing these hypotheses requires identifying PMCA and NCX proteins as expressed by RPE, and assaying their roles in RPE physiology. These are goals of this project. Experiments use freshly isolated RPE cells and tissue from human donor eyes and other animal sources. The approach is on 3 levels, molecular (AIM1), structural (AIM2), and biochemical/functional (AIM3). Molecular studies will identify PMCA and NCX isoforms expressed in RPE, and will sequence cDNAs that encode them. Structural studies using immunoEM will identify the membrane location (apical vs. basolateral) of these Ca2+ transport proteins in native RPE tissue. Biochemical studies will identify RPE PMCA and NCX proteins (immunoblotting), and examine their basic properties in freshly isolated RPE cells. Functional studies will examine transepithelial Ca2+ transport (ion flux studies), and regulation of [Ca2+]I (Ca2+ imaging) in freshly isolated RPE tissue. Ion substitution and selective inhibitors will be used to evaluate roles of PMCA and NCX activities in these processes. Proposed studies represent a new initiative in RPE cell biology. The molecular characterization of the PMCA and NCX has been well studied in erythrocytes and excitable cells, respectively. Proposed studies will extend findings to human RPE, and will contribute generally to the field of epithelial Ca2+ transport. Planned studies will identify the physiological significance of PMCA and NCX activities in RPE, and will help to identify ocular pathologies that could be caused by impaired PMCA or NCX function.